Regeneration of tooth germ using dental epithelial stem cells and dental papilla mesenchymal stem cells in rodent incisors

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Mouse incisors are regenerative tissues, which grow continuously throughout life. We found that in the teeth, Fgf-9 plays a role of maintenance of mesenchymal cells expressing Fgf-10 and that the stem cell niche of incisors are formed by the epithelial-mesenchymal interaction via the signaling of Fgf-9 and Fgf-10. Immunostaining showed that Fgf-9 was expressed in the basal epithelium, stellate reticulum and inner enamel epithelium in the apical bud, and the expression area underlay the mesenchyme expressing Fgf-10. Recombinant Fgf-9 protein stimulated the increase of number of mesenchymal cells in a concentration-dependent manner. Annexin V staining and whole mount in situ hybridyzation using organ culture showed that recombinant Fgf-9 protein inhibited the apoptosis of mesenchymal cells of apical end and maintained the expression of Fgf-10. However, we could not success to produce bioengineered tooth germ and to culture these stem cells in the presence of only these factors.Furthermore, we investigated the additionally factors in the culture medium and it was found that we needs FGF2 and EGF to culture the cells of the epithelial and mesenchymal stem cell compartment.Finally, we succeeded to produce the regenerative bioengineered teeth using the dental epithelial stem cells and the mesenchymal stem cells of mouse incisors by the combination of collagen sponge and Fgf2/Egf/Fgf9/Fgf10-releasing gelatin beads. Now, we proceed to study the technical methods controlling the morphology and size of bioengineered tooth by these factors.